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  • ElectroLigase®

    Description

    ElectroLigase™ combines T4 DNA ligase and an optimized, ready-to-use 2X reaction buffer containing a proprietary ligation enhancer and no PEG. This combination is specifically formulated to promote robust ligation of all types of DNA ends (blunt, sticky, TA). It is directly compatible, without desalting or purification, with electrocompetent cells used for transformation by electroporation. No thawing of the buffer is required as it maintains a liquid state during storage at -20°C*, thereby simplifying reaction set-up. By providing an optimized ratio of enzyme and buffer components, users are able to rapidly ligate all types of DNA ends applying a short incubation time at room temperature. Ligations for subcloning can be carried out in small volumes with low concentrations; allowing users to conserve precious DNA samples. These reactions can be pipetted directly, without purification or dilution, to transform many strains of electrocompetent E. coli**.

    * Freezers vary in their actual internal temperatures. Our testing demonstrates that the enzyme and buffer remain liquid at -20°C.

    **ElectroLigase is also compatible with chemically competent strains of E. coli. Performance is generally around 50% efficiency, when compared to the Blunt/TA Ligase Master Mix (NEB #M0367 ).

    M0369
    Ligation reactions containing equal amounts (20 ng vector and 3-fold molar excess of insert) of blunt- (A), T/A (B), or sticky-end (C) vector/insert pairs were set up using ElectroLigase and incubated for the times shown. After heat inactivation, 2 μl of each reaction were withdrawn and directly used to transform NEB 10-beta Electrocompetent E. coli (NEB #C3020). 50 μl aliquots of the outgrowth (diluted, in some cases) were plated onto selective plates and incubated overnight at 37°C. Colonies were counted, adjusted for plating dilution, and graphed.

    Product Source

    Purified from an E. coli strain containing a recombinant gene encoding T4 DNA Ligase.

    Reaction Volume Definition

    1X ElectroLigase Reaction Buffer with DNA substrates and 1 μl ElectroLigase in an 11 μl reaction volume incubated at 25°C for 30 minutes.

    Advantages and Features

    Applications

    • Vector construction
    • Linker ligation
    • Fragment assembly
    • Library construction
    • TA cloning

    Properties and Usage

    Storage Temperature

    -20°C

    Heat Inactivation

    65°C for 15 min

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Functional Test (Ligation and Transformation):
      The product is tested for ligation and transformation efficiency using DNA containing blunt or cohesive termini. Ligation products are transformed and must exceed specifications for efficiency (transformants/µg).

    Notes

    1. Usage Notes:

      Cells: Competent cells can vary by several logs in their competence. Perceived ligation efficiency directly correlates with the competence of the cells used for transformation. Always transform uncut vector as a control for comparison purposes.

      DNA: Purified DNA for ligations can be dissolved in dH2O (Milli-Q® water or equivalent is preferable); TE or other dilute buffers also work well. For optimum ligation, the amount of vector DNA should be 20–100 ng and the insert should be added at a 3-fold molar excess. For ligation volumes greater than 11 μl, increase the volume of ElectroLigase Reaction Buffer accordingly. Insert:vector ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.

      Time and Temperature: Most ligations performed using ElectroLigase reach an end point at 60 minutes or less when performed between 4–37°C. Incubation beyond this time provides no additional benefit. Our recommendation for a 25°C (room temperature) incubation was chosen after evaluation of performance at 4°C, 16°C, 25°C, and 37°C. Most conditions reached at least 50% performance within 30 minutes.

      Biology: Some DNA sequences are not easy to clone. Sequences that form structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. My transformations using ElectroLigase® reactions produced no colonies. What happened?
    2. Can ligation reactions set-up with ElectroLigase® be used for transformation of chemically competent cells?
    3. Do I need to dilute the ElectroLigase® reaction in order to use it to transform electrocompetent cells?
    4. Can ElectroLigase® reactions be incubated for longer than 30 minutes?
    5. Can I incubate an ElectroLigase® ligation reaction at a temperature other than 25°C?
    6. I used an ElectroLigase® reaction to transform electrocompetent cells and I experienced arcing of my sample. What happened?
    1. Ligation Protocol for Cloning with ElectroLigase® (M0369)
    2. Transformation Protocol (M0369)

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides